Clostridium perfringens type A is widely distributed (soil, sewage, intestinal tracts of humans and animals . . . ), and is a common causative agent of bacterial food poisoning outbreaks worldwide. The symptoms, predominantly diarrhea and abdominal pain, appear 6 to 24 hours after ingestion of contaminated food. Vomiting and fever are unusual. Death occurs occasionally among debilitated persons, particularly the elderly.
C. perfringens enterotoxin CPE which is produced during the sporulation phase has been shown to produce the symptoms associated with C. perfringens food poisoning. The illness is caused by the ingestion of food that contains larger number of vegetative enterotoxigenic C. perfringens (more than 10.sup.5 organisms per g). These bacteriae multiply and sporulate, releasing CPE into the intestine.
A C. perfringens count of more than 10.sup.6 /g in fecal samples of ill persons is indicative of C. perfringens food poisoning. In addition, CPE detection directly in fecal samples is a valuable method confirming the diagnosis.
The epidemiological investigations involve C. perfringens numeration in suspected foods. The characterization of the enterotoxigenic C. perfringens strains is not used routinely, since C. perfringens sporulates poorly in usual culture media.
Recently, CPE and phospholipase C gene sequences have been determined (VAN DAMME et al. 1989, Ant. Van Leeuwen 56, 181-190; TSO J. and SIEBEL, 1989. Infect. Immun. 57: 468-476, TITBALL et al. 1989 Infect. Immun. 57:367-376). The phospholipase C gene is located on a variable region of the chromosomal DNA in all C. perfringens toxinotypes whereas the distribution of CPE gene is restricted. Only 6% of the C. perfringens isolates from various origins showed the presence of CPE gene by DNA-DNA hybridization. This ratio is higher (59%) among C. perfringens strains isolated from confirmed outbreaks of food poisoning. In the standard methods, the enterotoxin detection by biological or immunological tests requires previously the sporulation of C. perfringens. Several specific medium and protocols for C. perfringens sporulation have been described, but they are time consuming and many C. perfringens strains do not sporulate or very poorly (DUCAN and STRONG, 1968. Appl. Microbiol. 16: 82; PHILIPS, 1986, Lett. Appl. Microbiol 3: 77-79), which impairs the CPE detection.
A method for the detection of C. perfringens by polymerase chain reaction (PCR) has been disclosed during the third Congress of the French Society of Microbiology (Apr. 21-24, 1992) through a poster of FACH et al.
The method consisted of amplification of the parts of the genes encoding the .alpha.-toxin, also called lecithinase, and the enterotoxin of C. perfringens by using oligonucleotidic primers which were choosen in these genes. However, the sequence of the primers used for carrying out this method was not disclosed and the sensitivity mentioned by the authors was low, i.e. from 500 to 5000 bacteriae per gram of feces.
Moreover, the results were obtained on feces artificially contaminated and not on feces from contaminated patients or no contaminated food.